JP6148708B2 - Rapid nucleic acid extraction method and apparatus - Google Patents

Description

  The present disclosure relates to nucleic acid extraction, and in particular to nucleic acid extraction via an extraction device or extraction apparatus.

  Nucleic acids are essential polymer macromolecules for all known organisms. Nucleic acids include deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) consisting of monomers called nucleotides. The most important macromolecules present in living organisms are nucleic acids and proteins. This is because nucleic acids and proteins serve to encode, transmit and express genetic information. An “extraction” step is required to test nucleic acids. Other names for “extraction” include “purification”, “separation”, “concentration” and the like. As a complex laboratory method for nucleic acid extraction, extraction through a chromatography column, separation by magnetic beads, and the like are known.

  According to an exemplary embodiment, a novel nucleic acid extraction method is provided that enables testing to be performed quickly, reliably, and in a portable manner. Various exemplary embodiments provide a novel platform for rapid nucleic acid extraction on-site without the need for expensive instruments and / or specialized laboratory environments.

  According to one embodiment, the binding step, the washing step, and the elution step of the nucleic acid material that accompany the extraction of the nucleic acid are incorporated in the extraction device, and the extraction device supplies a buffer and / or a solution necessary for nucleic acid extraction. Supply.

  According to an exemplary embodiment, the first container is configured to contain a nucleic acid extraction solution. The first container has a thin film at its lower end, and the thin film is configured to capture nucleic acids while allowing other components of the solution to permeate. The first container is disposed on a waste collection container configured to receive the lower end of the first container.

  Furthermore, according to this exemplary embodiment, a syringe can be inserted into the upper open end of the first container and comprises a capsule having a chamber filled with a wash buffer solution, the capsule being a plunger of the syringe. It is possible to move downward according to the operation. The capsule further includes a sealant provided over the upper opening and a spike attached to the lower end, the spike extending into the chamber and a first end extending out of the lower end of the capsule. A second end portion extending.

  In one embodiment, the configuration and position of the capsule inside the syringe is such that the nucleic acid solution in the first container is first pushed through the membrane as the capsule moves downward within the syringe, and then the spike is It is assumed that the capsule continues to move down while contacting the lower surface of the syringe and stops so that the spike buffer breaks or breaks the sealant, thereby releasing the wash buffer solution into the syringe. As the syringe moves further down, the wash buffer solution is pushed through the membrane, resulting in pure nucleic acid remaining on the membrane. The apparatus may further comprise a second syringe device configured to wash the thin film of the first container and elute RNA, DNA or other selected nucleic acid.

It is the schematic of each step of an exemplary nucleic acid solution preparation method. FIG. 2 is a side view showing additional steps and equipment used in an exemplary nucleic acid extraction process. It is a side view which shows the state which combined the apparatus of FIG. 2 is a side cross-sectional view of an exemplary wash buffer capsule. FIG. It is a sectional side view of the main-body component of the capsule shown in FIG. It is a top view of the capsule shown in FIG. It is a side view of the spike component of the capsule shown in FIG. FIG. 4 is a schematic side view showing the arrangement of the capsule shown in FIG. 4 in a syringe cooperating with the apparatus shown in FIGS. 2 and 3. FIG. 5 is a cross-sectional side view of the capsule shown in FIG. 4 in a position to release wash buffer. It is a figure which shows the process of obtaining a pure nucleic acid using a 2nd syringe. FIG. 6 is a side cross-sectional view of an alternative capsule embodiment. FIG. 5 shows an alternative capsule and capsule breaking mechanism. It is a figure which shows one Embodiment of a waste collection container. FIG. 6 is a side schematic view of an alternative embodiment. FIG. 6 is a side schematic view of another alternative embodiment.

  In an exemplary embodiment, referring to FIG. 1, a sample 11 or a solution 16 is supplied to a container 13 that has been previously filled with a nucleic acid production solution 14. Here, as an example of the sample 11 or the solution 16, for example, human saliva taken on a cotton swab, body fluid such as blood, sweat, or urine, skin, hair, plant sample, cell culture, microbial culture, soil, or Any other sample that may contain nucleic acids is included. The nucleic acid preparation solution 14 contains an appropriate pH buffer and other additives, and destroys the cells of the sample to release the nucleic acid. For example, the nucleic acid preparation solution 14 suppresses degradation of the nucleic acid by an enzyme and maintains the nucleic acid. is there. Additives include surfactants, chelating agents, salts, chaotropic reagents, solvents such as ethanol and isopropyl alcohol, acids, bases, and optionally, proteolytic enzymes such as DNA-degrading enzymes or RNA-degrading enzymes. it can. In one embodiment, the nucleic acid production solution 14 functions as a diluent and a pH adjuster. Thereafter, a mixing / shaking step is performed on the container 13, and as a result, a solution 15 for nucleic acid purification / extraction is generated.

As shown in FIG. 2, subsequently, a selected amount of the solution 15 is filled in the vial 17 serving as the first container. In one embodiment, the vial 17 can be composed of a cylindrical upper segment 19 and a conical tapered portion 21 that forms a cylindrical nozzle 23 having a smaller diameter than the cylindrical segment 19, and the nozzle 23 is a thin film (membrane). It is blocked at 25. The contents of the vial 17 may be referred to as a nucleic acid binding column. The thin film 25 serves to hold the nucleic acid in the solution 15, while unnecessary components are washed away through the thin film 25. In one embodiment, the thin film 25 can be silica-based. Numerous methods for producing the thin film 25 are known to those skilled in the art. In various embodiments, the thin film is silica (SiO ₂ ), silicon oxide (s), glass powder, alkyl silica, aluminum silicate (zeolite), activated silica having NH ₂ groups, or a mixture comprising any of the above. Can be configured.

  The apparatus shown in FIGS. 2 and 3 further includes a waste collection device 29, which is, for example, in one embodiment, a sealed plastic container having an opening 33 at the upper end and receiving the vial 17. Then, the vial 17 can be shaped to be held at a position where the vial 17 is disposed on the waste collection unit 35. Thus, in one embodiment, the vial 17 has an edge (lip) 18 along the top edge or rim. Such a lip 18 may be at a different location along the side of the vial 17 in various embodiments.

  Furthermore, according to this exemplary embodiment, a washing buffer container and a release capsule 41 are provided, one embodiment of which is shown in FIGS. The exemplary capsule 41 includes a body 43 (FIG. 5) that can be formed of plastic, and includes a hollow cylindrical chamber 64 and a first groove 47 and a second groove 49 formed on the outer periphery 51 of the body 43. With. Here, the first groove 47 and the second groove 49 are positioned in the vicinity of the upper and lower ends of the main body 43, respectively. A first O-ring 52 and a second O-ring 54 (FIG. 4) are provided in the groove 47 and the groove 49, respectively.

  In one embodiment, the capsule 41 further comprises a spike 53 that can be formed of plastic, and the spike 53 has an O-ring seal 55. One end of the spike 53 extends into the chamber 64, while the other end extends out of the chamber 64 and includes a forked tail 66. In the exemplary embodiment, spike 53 further comprises a tip 56.

  In one embodiment, the chamber 64 of the capsule 41 is filled with a wash buffer solution 67 (FIG. 8) and then sealed, for example by applying a thin metal foil 58 across the top opening 60. Stop. In various embodiments, the wash buffer solution comprises water, pH buffer, surfactant, chelating agent, salts, chaotropic reagents, solvents such as ethanol and isopropyl alcohol, acids, bases, and, optionally, a DNA-degrading enzyme or Proteolytic enzymes such as RNases can be included.

  Referring to FIG. 8, the shape and dimensions of the capsule 41 are raised and lowered in the barrel 61 of the syringe 59 with the O-rings 52 and 54 engaged with the inner side surface of the barrel 61 of the syringe 59 in a sealable manner. It turns out that it is shape | molded. In one embodiment, the end 62 of the syringe 59 is twisted into the top of the vial 17. In the exemplary embodiment, the sealing by the O-rings 52, 54 is a hermetic seal, and when the plunger of the syringe 59 or the drive piston 60 is actuated to push the capsule 41 downward, the action of air (air) pressure is applied. As a result, all the liquid in the vial 17 is pushed through the thin film 25 to the waste collection container 29 or forcedly discharged.

  When the plunger 60 of the syringe 59 is further pushed down, the fork tail portion 66 of the spike 53 of the capsule 41 contacts or abuts a part of the inner lower edge 63 of the syringe 59, as shown in FIG. Then, the operation of the O-ring 55 is stopped, and the spike 53 perforates the sealing material 58 provided over the entire upper portion of the capsule 41. By piercing the sealing material 58 in this manner, the washing buffer solution 67 is released, and then the washing buffer solution 67 is press-fitted into the end 68 or the opening 68 at the tip of the syringe 59 by the plunger 60, and then through the thin film 25. Pushed out. When the washing buffer solution 67 permeates the thin film 25, the nucleic acid held by the thin film 25 is washed, and the thin film 25 newly holds the substantially pure nucleic acid.

  As shown in FIG. 9, a second syringe 71 including a capsule 41 a configured similarly to the capsule 41 shown in FIGS. 4 to 7 is subsequently screwed into the vial 17 or attached in a sealable manner. Is used to further wash the pure nucleic acid with solution 75 and elute or wash the selected nucleic acid into a second container or tube 101. In one embodiment, if it is desired to release bound DNA, RNA, or other nucleic acid from the thin film, the final wash solution can be combined with water or a pH adjusting buffer to inhibit nucleic acid degradation or as known in the art. Optional additives to inhibit, such as chelating agents, may be included.

  It should be noted that various “spin column” commercial kits can also be used to work with the devices disclosed herein. This kit uses a spin column based on silica-based or similar absorption techniques. As an example, a spin column RNA / DNA purification kit manufactured by Qiagen N.V. of the Netherlands can be mentioned, and a preparation of the nucleic acid preparation solution 14, the washing buffer 67, and the eluate 75 can be obtained. Other manufacturers of similar kits include Zymo Research, Invitrogen / Life Sciences, Bio-Rad, Thermo Scientific, and Promega. Companies.

  The apparatus according to this exemplary embodiment can be combined with other rapid DNA / RNA detection methods such as disease diagnosis methods or clinical work / screening.

  The rapid nucleic acid extraction apparatus shown in FIGS. 1 to 14 can be the first step in a series of steps. First, DNA / RNA is extracted using a nucleic acid rapid extraction device. Next, 1 volume of the purified nucleic acid is transferred to a tube containing the reaction mixture of enzyme, primer, dNTP, salt, buffer, water using a pipette device or other suitable liquid transfer method. Then, the sample is placed under a predetermined series of thermal cycling conditions, or other amplification methods such as LAMP (Loop-mediated isothermal amplification) method are applied. Finally, the contents in the tube may be analyzed to determine the presence or absence of the target DNA / RNA.

  The thermal cycle described above can also be field mobile using a “handheld PCR machine” or the like. For on-site use, enzymes, primers, and other reaction mixtures (cocktails) should be adjusted to suit each target. The adjustment work can largely depend on experience and know-how, thereby optimizing the process. Similarly, the final re-detection can be on-site mobile.

  Reverse transcription PCR can be performed on Ebola and other RNA viruses / targets. Again, the success depends largely on the optimizations performed prior to generation, making the device sufficiently robust to withstand field use by untrained personnel in the field of biological sciences. To get.

  In the present exemplary embodiment, a simple and easily usable device with a relatively small number of parts is provided, but in other embodiments, a series of syringes may be used. For example, air pressure is applied by a first syringe to push out the nucleic acid solution through the thin film 25, and then the thin film 25 is washed with a washing buffer such as the washing buffer 67 using the second syringe (FIG. 4, Next, air pressure is applied by the third syringe, and for example, the eluate / buffer solution / aqueous solution indicated by reference numeral 75 in FIG. 9 is applied by the fourth syringe. Perform “washing”. In another embodiment, the capsule may be placed in a closed tube instead of a syringe, and air pressure is applied from a port of the tube under the capsule to push the nucleic acid solution indicated by reference numeral 15 through the thin film, for example. Thereafter, the capsule is pulled down under vacuum and the breaking mechanism is actuated to release, for example, a washing buffer indicated by reference numeral 67.

  For example, one side or both sides of the capsule denoted by reference numeral 41 can be sealed to accommodate the pre-filled contents. Capsule can also be filled from the open end with only one side sealed. Various embodiments may also include the use of multiple capsules simultaneously or sequentially, depending on the need and application. The syringes and capsules in each of the exemplary embodiments described above are disclosed in which the cross-sectional shape is circular, but in other embodiments, those having other cross-sectional shapes such as a square, a rectangle, and an ellipse are used. Please understand that you get.

  As shown in FIGS. 11, 13, and 14, for example, a capsule discharge shaft or spike indicated by reference numerals 103 and 113 for releasing the contents of the capsule indicated by reference numeral 102 is provided near the end of the syringe barrel 105. Or it can be attached to the body of the plunger 107 and then each shaft or each spike can be driven downward to puncture or break the capsule 102.

  The capsule orientation can also be reversed. In that case, for example, a spike indicated by reference numeral 53 is located at the upper part and faces a plunger indicated by reference numeral 60, for example, and the perforated end 55 faces downward toward the bottom surface of the syringe. In another embodiment 90 of the capsule shown in FIG. 10, the capsule is closed with a disc or plug 91 attached to the upper end of the shaft 93. Here, the structure of the other components of the capsule is as shown in FIGS.

  Instead of the O-ring used in each exemplary embodiment, for example, a dynamic sealing technique such as a soft plastic ridge, or alternatively, for example, 109, 110 (FIG. 14) provided before and after the capsule The spring shown can be used to adjust capsule positioning and release timing.

  Thus, one of ordinary skill in the art appreciates that various modifications and changes can be made to the preferred embodiments described herein without departing from the scope and spirit of the invention. Therefore, it is to be understood that, within the scope of the appended claims, the invention may be practiced other than as specifically described herein.

Claims (26)

Producing a breakable capsule containing a wash buffer;
Inserting the capsule into a syringe device, inserting the capsule such that the capsule is located above a lower end of the syringe device and below a plunger of the syringe device;
The syringe device is a step of inserting a lower end portion of the syringe device into a container containing a solution containing nucleic acid, the container further comprising a thin film configured to capture the nucleic acid. Inserting the lower end of
The step of pushing in the plunger of the syringe device is to push out the solution through the thin film and hold the nucleic acid by the thin film while allowing unnecessary components in the solution to pass through the thin film. Steps,
Thereafter, breaking the capsule to release the washing buffer, and further advancing the plunger of the syringe device, the washing buffer being out of the lower end of the syringe device through the thin film Advancing, allowing the liquid to drain while leaving pure nucleic acid on the membrane.   The method of claim 1, wherein the solution is pushed through the membrane by air pressure generated by pushing the plunger.   3. The method of claim 1 or 2, comprising eluting the sorted nucleic acid using a second syringe device configured to further wash the thin film.   The sorted nucleic acid comprises one of RNA, DNA, complementary DNA, complementary RNA, modified DNA, modified RNA, transfer RNA, ribosomal RNA, tmRNA, PNA, LNA, GNA, or TNA The method of claim 3.   The breakable capsule comprises a three-dimensional hollow body part that can be broken so as to release the washing buffer, and a material attached to the three-dimensional hollow body part. The method according to any one of the above.   The method according to claim 5, wherein the material is a thin metal foil, and the capsule is broken by perforating the thin foil. A syringe device using a first container having an upper end with an opening and a lower end closed with a thin film configured to hold a nucleic acid, and further including a barrel and a plunger movably attached in the barrel A rapid nucleic acid extraction method using
Producing a wash buffer release capsule containing the wash buffer;
Inserting the wash buffer release capsule into the syringe device;
A step of pre-filling a second container with a nucleic acid preparation solution, the nucleic acid preparation solution being selected to destroy the cells of the live sample to release the nucleic acid and to maintain the nucleic acid; Filling, and
Placing a raw sample containing nucleic acid into the pre-filled second container, stirring the contents of the second container to produce a treated nucleic acid extraction solution;
Filling the first container with a selected amount of the treated nucleic acid solution;
Inserting the syringe device into the first container;
Pushing the plunger of the syringe device downward, by first extruding the treated nucleic acid solution through the membrane by air pressure to capture the nucleic acid on the membrane, while the other components of the treated nucleic acid solution are Passing the thin film through;
Then releasing the wash buffer from the wash buffer release capsule at a position below the plunger;
A step of advancing the plunger downward, wherein the washing buffer is drained through the membrane to leave pure nucleic acid on the membrane.   The method according to claim 7, wherein the raw sample includes a solid substance.   The method according to claim 7, wherein the raw sample is a liquid.   The method according to any one of claims 7 to 9, wherein the sample is mixed with the nucleic acid preparation solution by stirring the contents of the second container.   The method according to any one of claims 7 to 9, characterized in that the contents of the second container are agitated by using a shaking step.   The method according to any one of claims 7 to 11, further comprising the step of further washing the thin film using a second syringe device to elute the selected nucleic acid.   The selected nucleic acid includes one of RNA, DNA, complementary DNA, complementary RNA, modified DNA, modified RNA, transfer RNA, ribosomal RNA, tmRNA, PNA, LNA, GNA, or TNA. 13. A method according to claim 12, characterized.   14. The capsule according to any one of claims 7 to 13, wherein the capsule comprises a three-dimensional hollow body part that can be broken so as to release the washing buffer, and a material attached to the three-dimensional hollow body part. The method according to item. Producing a breakable capsule containing a wash buffer;
Inserting the capsule into a syringe device;
Inserting the syringe device into a container comprising a nucleic acid solution and a thin film configured to capture the nucleic acid;
Pushing the plunger of the syringe device, first pushing the solution through the membrane; pushing the plunger;
Then breaking the capsule to release the wash buffer;
And advancing the plunger, wherein the advancing step drains the wash buffer through the membrane. A method for nucleic acid extraction using a syringe device comprising a breakable capsule containing a wash buffer located above a lower end of the syringe device and below a plunger of the syringe device comprising:
Inserting the lower end of the syringe device into a container containing a solution containing nucleic acid, the container further comprising a thin film configured to capture nucleic acid; When,
The step of pushing in the plunger of the syringe device, first pushing out the solution through the thin film and holding the nucleic acid by the thin film while allowing unnecessary components in the solution to pass through the thin film. When,
Thereafter, breaking the capsule to release the washing buffer, and further advancing the plunger of the syringe device, the washing buffer being out of the lower end of the syringe device through the thin film Advancing, allowing the liquid to drain while leaving pure nucleic acid on the membrane. By pushing the plunger by the generated air pressure, wherein the solution is extruded through the membrane The method of claim 16.   18. The method according to claim 16 or 17, comprising eluting the sorted nucleic acid using a second syringe device configured to further wash the thin film.   The selected nucleic acid includes one of RNA, DNA, complementary DNA, complementary RNA, modified DNA, modified RNA, transfer RNA, ribosomal RNA, tmRNA, PNA, LNA, GNA, or TNA. 19. A method according to claim 18, characterized.   20. The breakable capsule comprises a solid hollow body part that can be broken so as to release the washing buffer, and a material attached to the solid hollow body part. The method according to any one of the above. A first container configured to contain a nucleic acid extraction solution and having a thin film located at a lower end thereof, wherein the thin film captures nucleic acid while allowing other components of the solution to pass therethrough. A first container, characterized by comprising:
A syringe that can be inserted into the upper open end of the first container, the syringe comprising a capsule that can be moved downward in response to the operation of a plunger of the syringe, and the capsule is a spike attached to the lower end of the syringe. The spike has a first end extending out of the lower end of the capsule, the capsule further comprising a sealing material provided over the entire upper opening thereof, Further comprises a syringe characterized by containing a buffer solution;
The configuration and position of the capsule inside the syringe are such that when the capsule moves downward inside the syringe, the nucleic acid solution in the first container is first pushed out through the thin film, and then the spike is A nucleic acid extraction apparatus characterized in that the buffer solution is released into the first container by contacting the lower surface of the syringe and puncturing the sealing material. The nucleic acid extraction apparatus according to claim 21, further comprising:
The buffer is further moved downward to push the buffer solution through the membrane, resulting in pure nucleic acid remaining on the membrane,
Nucleic acid extraction device. The nucleic acid extraction device according to claim 21, further comprising a second syringe device configured to further wash the thin film and elute the selected nucleic acid.
Nucleic acid extraction device. The nucleic acid extraction apparatus according to claim 23,
The selected nucleic acid includes one of RNA, DNA, complementary DNA, complementary RNA, modified DNA, modified RNA, transfer RNA, ribosomal RNA, tmRNA, PNA, LNA, GNA, or TNA. Features
Nucleic acid extraction device. A container containing a solution containing nucleic acid, and further comprising a thin film configured to capture nucleic acid;
A breakable capsule containing a wash buffer;
A ruptured part that breaks the capsule and releases the washing buffer;
A syringe device, wherein the capsule is positioned above a lower end portion of the syringe device and below a plunger of the syringe device, and the lower end portion of the syringe device is shaped to fit into the open end of the container. A syringe device,
A nucleic acid extraction apparatus comprising: a plunger of the syringe device that can be pushed down, and first pushes out the solution through the thin film so that unnecessary components of the solution pass through the thin film while the thin film holds the nucleic acid. There,
Pure nucleic acid on the thin film by breaking the capsule into the breakable part, releasing the washing buffer, and then draining the washing buffer out of the lower end of the syringe device through the thin film The device is characterized in that the plunger is further depressible to leave A breakable capsule containing a wash buffer ;
A syringe device comprising the capsule disposed below the plunger;
A sealing mechanism provided in conjunction with the capsule , the sealing mechanism capable of moving with respect to the syringe device simultaneously with sealing between the capsule and the syringe device ;
A breaking mechanism for breaking the capsule;
A container containing a nucleic acid solution, comprising a thin film arranged and configured to supplement nucleic acid;
A device comprising:
The capsule, the syringe, the plunger, the sealing mechanism, and the breaking mechanism apply air pressure from the syringe to the container, and then supply the cleaning buffer contained in the capsule from the syringe to the container. An apparatus characterized by being configured as described above.